Key Variables in ICS Assays Holden T. Maecker

The original BD ICS assay protocol 1 ml whole blood + Ag + CD28 + CD49d + Brefeldin A + FACSLyse, 10 min + EDTA/PBS 2h 4h 15 min Centrifuge, aliquot into staining tubes or freeze Wash Wash, fix, analyze CD69 PE anti-TNF FITC FACS Perm, 10 min mAb staining, 30-60 min 3- or 4-color Flow cytometry

96-well plate ICS protocol with lyophilized reagents PBMC or whole blood Plate with lyophilized Ag+BFA 6h @ 37oC, EDTA, FACS Lyse, FACS Perm 2 Plate with lyophilized Ab to flow cytometer

What are the key variables? Stimulation PBMC vs. whole blood Resting PBMC before activation Activation/staining in plates vs. tubes Costimulatory Abs (CD28+CD49d) Stimulation time Brefeldin A and/or monensin Processing and staining Use of EDTA or not Method of fixation/permeabilization Freezing of cells post-fixation Surface vs. intracellular staining for CD3, CD4, CD8, etc. Fluorochromes and fixation (APC-Cy7, AmCyan) Staining time, temperature, and agitation Number of washes (post-fix and post-stain) Acquisition and analysis Number of events to collect Gating Interpretation of results, precision, comparisons with other assays

Effect of overnight rest on cryo PBMC % IFNg+ MFI of IFNg+ 3 750 2 500 1 250 100 10000 1000000 100 10000 1000000 Reciprocal Dilution Reciprocal Dilution No rest, 6 h peptide stimulation Overnight rest, 6 h peptide stimulation

Correlation of tube vs. plate ICS A. 96-well whole blood B. 96-well PBMC CD4 CD8 CD4 CD8 1.25 2.5 4 4 1.00 2.0 3 3 Peptide mix % IFNg+ cells 0.75 1.5 2 2 0.50 r 2 = 0.95 1.0 r 2 =0.87 r 2 = 0.96 r 2 = 0.73 p<0.0001 p<0.0001 p < 0.0001 p = 0.0002 0.25 1 1 slope 0.94 0.5 slope 0.88 slope = 1.2 slope = 1.1 0.00 0.0 plate 0.00 0.25 0.50 0.75 1.00 1.25 0.0 0.5 1.0 1.5 2.0 2.5 1 2 3 4 1 2 3 4 0.0 2.5 5.0 7.5 10.0 2 4 6 8 10 12 14 10 20 10 20 30 % IFNg+ cells SEB 2 2 r = 0.94 r 2 =0.95 r = 0.74 r 2 = 0.79 p<0.0001 p = 0.0002 p < 0.0001 p<0.0001 slope = 0.84 slope 0.91 slope 0.93 slope = 0.78 % IFNg+ cells % IFNg+ cells % IFNg+ cells % IFNg+ cells tube

Similar IFNg MFI in tubes vs. plates A. 96-well whole blood B. 96-well PBMC C. 24-well whole blood (HIV+) CD4 CD8 1 2 3 CD4 CD8 1 2 3 CD4 CD8 1 2 3 t u b e p l a t e IFNg mean fluorescence

Cell recovery in tubes vs. plates 1 7 5 % CD3+ cell recovery 5 2 5 Tube Tube 96-well 24-well 96-well Whole Blood PBMC

Effect of costimulatory Abs (CD28+49d)

Monensin vs. Brefeldin Summary Brefeldin A preferred (10 mg/mL): TNFa, IFNg, IL-2, IL-4, IL-12 When combining any of the above with CD107, TGFb, or IL-10: 5 mg/mL monensin + 5 mg/mL brefeldin A

What are the key variables? Stimulation PBMC vs. whole blood Resting PBMC before activation Activation/staining in plates vs. tubes Costimulatory Abs (CD28+CD49d) Stimulation time Brefeldin A and/or monensin Processing and staining Use of EDTA or not Method of fixation/permeabilization Freezing of cells post-fixation Surface vs. intracellular staining for CD3, CD4, CD8, etc. Fluorochromes and fixation (APC-Cy7, AmCyan) Staining time, temperature, and agitation Number of washes (post-fix and post-stain) Acquisition and analysis Number of events to collect Gating Interpretation of results, precision, comparisons with other assays

EDTA addition in plates and tubes

Fix/perm comparison: monocyte IL-12

Fix/perm comparison: T cell TGFb BD FACS Lysing Solution BD Cytofix/Cytoperm BD FACS Permeabilizing Sol’n 2 BD Perm Wash TGFb PE TGFb PE TGFb PE TGFb PE

TGFb staining and activation media TGFb PE TGFb PE TGFb PE

Freezing activated cells in FACS Lyse

Surface vs. IC staining for CD3 SEB stimulated PBMCs surface stain intracellular stain CD3 Alexa 700 Clone SP34-2 @0.8mg/test

Surface vs. IC staining for CD4 & CD8 surface stain intracellular stain SEB stimulated PBMCs CD8 APC-hilyte 750 @125ng CD4 AmCyan @ 60ng CD8 Qdot 605 @ 0.25ul CD4 AmCyan @ 60ng

All events CD3+ gate B CD8 APC-Cy7 Side Light Scatter C A CD3 Pacific Blue CD4 AmCyan CD4- CD8+ gate CD4+ CD8- gate IFNg FITC IFNg FITC IL-2 PE IL-2 PE CD8+ IFNg+ CD8+ IFNg+ IL-2+ CD4+ IFNg+ CD4+ IFNg+ IL-2+ Side Light Scatter Side Light Scatter Side Light Scatter Side Light Scatter CD27 APC CD27 APC CD27 APC CD27 APC CD45RA PE-Cy7 CD45RA PE-Cy7 CD45RA PE-Cy7 CD45RA PE-Cy7 CD45RA PE-Cy7 CD45RA PE-Cy7 CD45RA PE-Cy7 CD45RA PE-Cy7 CD28 PerCP-Cy5.5 CD28 PerCP-Cy5.5 CD28 PerCP-Cy5.5 CD28 PerCP-Cy5.5 CD28 PerCP-Cy5.5 CD28 PerCP-Cy5.5 CD28 PerCP-Cy5.5 CD28 PerCP-Cy5.5

Degradation of APC-Cy7 on stained cells In BDSF

BDSF and AmCyan don’t mix

IC staining time, volume, and mixing

What are the key variables? Stimulation PBMC vs. whole blood Resting PBMC before activation Activation/staining in plates vs. tubes Costimulatory Abs (CD28+CD49d) Stimulation time Brefeldin A and/or monensin Processing and staining Use of EDTA or not Method of fixation/permeabilization Freezing of cells post-fixation Surface vs. intracellular staining for CD3, CD4, CD8, etc. Fluorochromes and fixation (APC-Cy7, AmCyan) Staining time, temperature, and agitation Number of washes (post-fix and post-stain) Acquisition and analysis Number of events to collect Gating Interpretation of results, precision, comparisons with other assays

Number of events to acquire CALCULATING SAMPLE SIZE FOR RARE EVENT ANALYSIS Enter the estimated values in the grey fields: % bkgd= 0.1 p(av)= 0.0015 %pos= 0.2 delta= 0.001   For power of 90%, p<0.05, N= 25761 For power of 99%, p<0.005, 71592 For power of 80%, p<0.05, 18572 _______________________________________________________________ ________# events to collect__________ % bkgd lowest % positive 90% power, p<0.05 99% power, p<0.005 0.01 0.02 260,000 720,000 0.05 32,000 90,000 12,000 67,000 190,000 16,000 45,000 0.03 170,000 480,000 23,000 63,000 0.04 33,000 93,000 52,000 140,000 0.06 86,000 240,000 0.07 160,000 450,000 0.08 17,000 46,000 26,000 72,000

Importance of gating dim cells

Multi-site lyophilized control cell data Individual (Manual) Analysis Cocltail 1 CD4 Cocktail 1 CD8 Cocktail 2 CD4 Cocktail 3 CD8 10 20 30 32% 29% 7% 14% % cytokine+ cells Central (Automated) Analysis 7% 3% 3% 3%

Gating Manual analysis Batch analysis with dynamic gates 0.55% 0.56% 200 400 600 800 1000 Forward Scatter R1 10 1 2 3 4 CD3 APC R2 200 400 600 800 1000 Forward Scatter R1 10 1 2 3 4 R3 R2 CD3 APC ungated ungated ungated gated on R1 Side Scatter Side Scatter Side Scatter Side Scatter 10 1 2 3 4 CD3 APC R3 10 1 2 3 4 CD3 APC R4 R5 gated on R1 gated on R1&R2 &R3 ungated gated on R1&R3 &R5 R4 0.55% R6 0.56% CD8 PerCP-Cy5.5 CD69 PE CD8 PerCP-Cy5.5 CD69 PE R7 10 10 1 10 2 10 3 10 4 10 10 1 10 2 10 3 10 4 IFNg FITC IFNg FITC

What are the key variables? Stimulation PBMC vs. whole blood Resting PBMC before activation Activation/staining in plates vs. tubes Costimulatory Abs (CD28+CD49d) Stimulation time Brefeldin A and/or monensin Processing and staining Use of EDTA or not Method of fixation/permeabilization Freezing of cells post-fixation Surface vs. intracellular staining for CD3, CD4, CD8, etc. Fluorochromes and fixation (APC-Cy7, AmCyan) Staining time, temperature, and agitation Number of washes (post-fix and post-stain) Acquisition and analysis Number of events to collect Gating Interpretation of results, precision, comparisons with other assays

Holden’s rules for ICS (and life) Validate your assay, but don’t validate that the earth is round take advantage of others’ data if it’s sound Know the inherent variability in your assay don’t talk about significant differences unless you run at least triplicate samples Avoid comparing apples and oranges any changes to the assay can produce variability, so keep protocols constant and run samples in parallel whenever possible

Acknowledgements Laurel Nomura Smita Ghanekar Brinda Emu Maria Jaimes Margaret Inokuma Sonny Bhatia Joyce Ruitenberg Maria Suni Jack Dunne Skip Maino Brinda Emu Rebecca Hoh Steve Deeks Jeff Martin Mike McCune Doug Nixon